Project Summary/Abstract Glioblastoma multiforme (GBM) is the most prevalent and lethal primary brain tumor, with over 11,000 cases estimated to be diagnosed in 2016. Despite advanced treatments, median survival time for GBM patients is less than 15 months, mostly due to the high rate of tumor recurrence (95% of patients). Tumor-initiating cells (TICs) are thought to be responsible for recurrence, because they are more resistant to radio- and chemotherapy than other cancer cells and can re-populate the tumor. Thus, selective targeting of TICs by hitting their unique features could prevent recurrence. Self-renewal ability is one of the distinctive characteristics of TICs, and it is controlled by few genes. BMI1, a protein part of the chromatin remodeler complex PRC1, has been proven to sustain TICs self-renewal in GBM. However, our current knowledge about BMI1 post-translational regulation is inadequate. Hence, determining the pathways regulating BMI1 stability in GBM could provide important clues for the design of therapeutic strategies toward TICs and, ultimately, increase GBM patients? survival. The E3 ubiquitin ligase Fbw7 has been shown to regulate stability of many proteins driving tumor progression, such as c-Myc, Cyclin E and Notch. Fbw7 initiates protein degradation by binding to a specific consensus phosphodegron (CPD) sequence on its target proteins. Interestingly, we discovered that BMI1 contains this sequence. Our central hypothesis is that Fbw7 binds to BMI1 and promotes its degradation, and that Fbw7-mediated regulation of BMI1 stability impairs self-renewal ability of TICs in GBM. This research is using mechanistic studies to examine if Fbw7 promotes BMI1 proteasomal degradation, by assessing the effects of loss of Fbw7 and impaired proteasome function on BMI1 protein levels. Moreover, we will test the role of CPD sequence and its phosphorylation status on BMI1/Fbw7 interaction, through co- immunoprecipitation experiments, ubiquitination assays, and in vitro binding assays. Furthermore, as demonstrated for other Fbw7 targets, we will evaluate the role of GSK3? in priming BMI1 for degradation by inhibiting this kinase or making it constitutively active. Because of the known primary role of BMI1 in GBM TICs, this research aims to demonstrate that Fbw7-mediated BMI1 degradation impairs TICs? self-renewal ability in GBM. We will test the functional effect of Fbw7 expression on patient-derived GBM neurospheres in terms of cell self-renewal, cell differentiation, tumor formation and radio-/chemoresistance. Both in vitro assays (differentiation, colony formation, cell viability and apoptosis assays) and in vivo assays (orthotopic serial transplantation of patient-derived GBM neurospheres into immunodeficient mice before and after therapeutic intervention) will serve the scope. These proposed studies will uncover the mechanisms regulating BMI1 protein stability, thus helping the design of therapeutic strategies selectively targeting TICs in GBM.